Non-maltogenic exoamylases and their use in retarding retrogradation of starch

ABSTRACT

The present invention relates to the use of non-maltogenic exoamylases of retarding the detrimental retrogradation of starch. Furthermore, the invention relates to a novel non-maltogenic exoamylase from  Bacillus Clausii.

This is a divisional application of U.S. application Ser. No.09/647,504, Feb. 28, 2001, now U.S. Pat. No. 6,667,065, which is anational stage application of PCT/IB99/00649, filed Mar. 30, 1999, whichclaims priority to Denmark Application No. DK 0457/98, filed Apr. 1,1998.

FIELD OF THE PRESENT INVENTION

The present invention relates to proteins, especially proteins that arecapable of degrading starch.

In particular, the present invention relates to the use of proteins thatare capable of retarding the detrimental retrogradation of starch.

Detrimental retrogradation processes, such as staling, typically occurafter the heating and cooling of starch media, in particular aqueousstarch suspensions, and are due to transformation of gelatinised starchto an increasingly ordered state.

More in particular, the present invention relates to the use of proteinsthat are capable of retarding the detrimental retrogradation ofamylopectin.

More in particular, the present invention relates to the use of proteinsto prepare baked bread products, as well as to the baked bread productsthemselves.

More in particular, the present invention relates to retardation ofstaling in baked farinaceous bread products.

More specifically the present invention relates to a process for makinga baked farinaceous bread product having retarded or reduced staling,comprising adding a non-maltogenic exoamylase to the bread dough.

The present invention also relates to an improver composition for doughand baked farinaceous bread products comprising a non-maltogenicexoamylase.

BACKGROUND OF THE PRESENT INVENTION

Starch comprises amylopectin and amylose. Amylopectin is a highlybranched carbohydrate polymer with short α-(1→4)-D-glucan chains whichare joined together at branch points through α-(1→6) linkages forming abranched and bushlike structure. On average, there is one branch pointfor every 20-25 α-(1→4) linked glucose residues. In contrast, amylose isa linear structure mainly consisting of unbranched α-(1→4)-D-glucanunits. Typically, starches contain about 75% amylopectin molecules andabout 25% amylose molecules.

More specifically, linear malto-oligosaccharides are composed of 2-10units of α-D-glucopyranose linked by an α-(1→4) bond. Due to theirproperties such as low sweetness, high waterholding capacity, andprevention of sucrose crystallisation [1] these compounds have potentialapplications in the food industry. The preparation ofmalto-oligosaccharides with a degree of polymerisation (DP) above 3(i.e. DP>3) in larger amounts is however tedious and expensive.

As background information, DP1=glucose, DP2=maltose, DP3=maltotriose,DP4=maltotetraose, DP5=maltopentaose, DP6=maltohexaose,DP7=maltoheptaose, DP8=maltooctaose, DP9=maltononaose, andDP10=maltodecaose.

The discovery of microbial enzymes, which produce malto-oligosaccharidesof a specific length could allow the production of larger amounts ofthese oligosaccharides [2].

Amylases are starch-degrading enzymes, classified as hydrolases, whichcleave α-D-(1→4) O-glycosidic linkages in starch. Generally, α-amylases(E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase) are defined asendo-acting enzymes cleaving α-D-(1→4) O-glycosidic linkages within thestarch molecule in a random fashion [3]. In contrast, the exo-actingamylolytic enzymes, such as β-amylases (E.C. 3.2.1.2, α-D-(1→4)-glucanmaltohydrolase), and some product-specific amylases cleave the starchmolecule from the non-reducing end of the substrate [4]. β-Amylases,α-glucosidases (E.C. 3.2.1.20, α-D-glucoside glucohydrolase),glucoamylase (E.C. 3.2.1.3, α-D-(1→4)-glucan glucohydrolase), andproduct-specific amylases can produce malto-oligosaccharides of aspecific length from starch.

Several amylases producing malto-oligosaccharides of a specific DP havebeen identified previously including maltohexaose-producing amylasesfrom Klebsiella pneumonia [5, 6], Bacillus subtilis [7], B. circulansG-6 [8], B. circulans F-2 [9, 10], and B. caldovelox [11, 12].Maltopentaose-producing amylases have been detected in B. licheniformis584 [13] and Pseudomonas spp. [14, 15]. Furthermore,maltotetraose-producing amylases have been reported from Pseudomonasstutzeri NRRL B-3389 [16, 17], Bacillus sp. MG-4 [18] and Pseudomonassp. IMD353 [19] and maltotriose-producing amylases from Streptomycesgriseus NA-468 [20] and B. subtilis [21].

EP-B1-298,645 describes a process for preparing exo-maltotetraohydrolaseof Pseudomonas stutzeri or P. saccharophila using genetic engineeringtechniques.

U.S. Pat. No. 5,204,254 describes a native and a genetically modifiedexo-maltopentao-hydrolase of an alkalophilic bacterium (DSM 5853).

Very few product-specific amylases active at high pH have beenidentified. Examples of those that have been identified include amylasesfrom Bacillus sp. H-167 producing maltohexaose [22, 23], from abacterial isolate (163-26, DSM 5853) producing maltopentaose [24], fromBacillus sp. IMD370 producing maltotetraose and smallermalto-oligosaccharides [25], and from Bacillus sp. GM 8901 thatinitially produced maltohexaose from starch which was converted tomaltotetraose during extended hydrolysis periods [26].

Starch granules heated in the presence of water undergo anorder-disorder phase transition called gelatinization, where liquid istaken up by the swelling granules. Gelatinization temperatures vary fordifferent starches and depend for the native, unmodified starches ontheir biological source.

Cooling converts the gelatinised phase into a viscoelastic paste orelastic gel, depending on the starch concentration. During this process,amylose and amylopectin chains reassociate to form a more orderedstructure. With time, more associations are formed and they become evenmore ordered. It is believed that associations of amylopectin chains DP15-20 lead to a thermoreversible, quasi-crystalline structure.

In consequence of detrimental retrogradation, the water-holding capacityof the paste or gel system is changed with important implications on thegel texture and dietary properties.

It is known that the quality of baked bread products graduallydeteriorates during storage. The crumb loses softness and elasticity andbecomes firm and crumbly. This so-called staling is primarily due to thedetrimental retrogradation of starch, which is understood to be atransition of the starch gelatinised during baking from an amorphousstate to a quasi crystalline state. The increase in crumb firmness isoften used as a measure of the staling process of bread.

Upon cooling of freshly baked bread the amylose fraction, within hours,retrogrades to develop a network. This process is beneficial in that itcreates a desirable crumb structure with a low degree of firmness andimproved slicing properties. More gradually crystallisation ofamylopectin takes place within the gelatinised starch granules duringthe days after baking. In this process amylopectin is believed toreinforce the amylose network in which the starch granules are embedded.This reinforcement leads to increased firmness of the bead crumb. Thisreinforcement is one of the main causes of bread staling.

The rate of detrimental retrogradation or crystallisation of amylopectindepends on the length of the side chains of amylopectin. In accordancewith this, cereal amylopectin retrogrades at a slower rate thanamylopectin from pea or potato, which has a longer average chain lengththan cereal amylopectin.

This is supported by observations from amylopectin gel systems thatamylopectin with average chain length of DP, i.e. degree ofpolymerisation, ≦11 do not crystallise at all. Furthermore the presenceof very short chains of DP 6-9 seems to inhibit the crystallisation ofsurrounding longer side chains probably because of steric hindrance.Thereby these short chains seem to have a strong anti-detrimentalretrogradation effect. In accordance with this, amylopectinretrogradation is directly proportional to the mole fraction of sidechains with DP 14-24 and inversely proportional to the mole fraction ofside chains with DP 6-9.

In wheat and other cereals the external side chains in amylopectin arein the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectinside chains can markedly reduce their crystallisation tendencies.

It is known in the art to retard the staling of bread by usingglucogenic and maltogenic exo-amylases—such as amylogycosidases whichhydrolyse starch by releasing glucose—and maltogenic exoamylases orβ-amylases—which hydrolyse starch by releasing maltose from thenon-reducing chain ends.

In this respect, Jakubczyk et al. (Zesz. Nauk. Sck. GI.Gospod Wiejsk.Warzawie, Technol. Reino-Spozyw, 1973, 223-235) reported thatamyloglucosidase can retard staling of bread baked on wheat flour.

JP-62-79745 and JP-62-79746 state that the use of a β-amylase producedby Bacillus stearothermophilus and Bacillus megaterium, respectively maybe effective in retarding staling of starchy foods, including bread.

EP-A412,607 discloses a process for the production of a bread producthaving retarded staling properties by the addition to the dough of athermostable exoamylase, which is not inactivated before gelatinization.Only amyloglycosidases and β-amylases are listed as suitable exoamylasesto be used. The exoamylase is in an amount which is able to modifyselectively the crystallisation properties of the amylopectin componentduring baking by splitting off glucose or maltose from the non-reducingends of amylose and amylopectin. According to EP-A-412,607, theexoamylase selectively reduces the crystallisation properties ofamylopectin, without substantially effecting the crystallisationproperties of amylose.

EP-A-494,233 discloses the use of a maltogenic exoamylase to releasemaltose in the α-configuration and which is not inactivated beforegelatinization in a process for the production of a baked product havingretarded staling properties. Only a maltogenic α-amylase from Bacillusstrain NCIB 11837 is specifically disclosed. Apparently, the maltogenicexoamylase hydrolyses (1→4)-α-glucosidic linkages in starch (and relatedpolysaccharides) by removing a-maltose units from the non-reducing endsof the polysaccharide chains in a stepwise manner.

Thus, the prior art teaches that certain glucogenic exoamylases andmaltogenic exoamylases can provide an antistaling effect by selectivelyreducing the detrimental retrogradation tendencies of amylopectinthrough shortening of the amylopectin side chains.

Nevertheless, there is still a need to provide different and effective,preferably more effective, means for retarding the detrimentalretrogradation, such as retarding the staling, of starch products, inparticular baked products, more in particular bread products.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention provides a process for making a starch productthat has a retarded detrimental retrogradation property.

The present invention also provides enzymes that are useful in theprocess of the present invention.

The enzymes of the present invention are amylase enzymes. More inparticular, the enzymes of the present invention are non-maltogenicexoamylase enzymes.

It is to be noted that non-maltogenic exoamylases have not hitherto beenused to retard the detrimental retrogradation of starch products, letalone to retard staling in baked products.

Thus, according to a first aspect of the present invention there isprovided a process for making a starch product comprising adding to astarch medium a non-maltogenic exoamylase that is capable of hydrolysingstarch by cleaving off linear maltooligosaccharides, predominantlyconsisting of from four to eight D-glucopyranosyl units, from thenon-reducing ends of the side chains of amylopectin.

Addition of the non-maltogenic exoamylase to the starch medium may occurduring and/or after heating of the starch product.

Thus, according to a second aspect of the present invention there isprovided a baked product obtained by the process according to thepresent invention.

Thus, according to a third aspect of the present invention there isprovided an improver composition for a dough; wherein the compositioncomprises a non-maltogenic exoamylase, and at least one further doughingredient or dough additive.

Thus, according to a fourth aspect of the present invention there isprovided the use of a non-maltogenic exoamylase in a starch product toretard the detrimental retrogradation of the starch product.

Thus, according to a fifth aspect of the present invention there isprovided a novel non-maltogenic exoamylase.

These and other aspects of the present invention are presented in theacompanying claims. In addition, these and other aspects of the presentinvention, as well as preferred aspects thereof, are presented anddicussed below.

General Definitions

Thus, the present invention relates to the use of proteins that arecapable of retarding the detrimental retrogradation of starch media, inparticular starch gels.

In one preferred aspect, the present invention relates to the use ofproteins that are capable of retarding the staling of starch.

In another aspect, the present invention relates to the use of proteinsthat are capable of retarding the detrimental retrogradation of starchmedia, such as starch gels.

In accordance with the present invention, the term “starch” means starchper se or a component thereof, especially amylopectin.

In accordance with the present invention, the term “starch medium” meansany suitable medium comprising starch.

The term “starch product” means any product that contains or is based onor is derived from starch.

Preferably, the starch product contains or is based on or is derivedfrom starch obtained from wheat flour.

The term “wheat flour” as used herein is a synonym for the finely-groundmeal of wheat or other grain. Preferably, however, the term means flourobtained from wheat per se and not from another grain. Thus, and unlessotherwise expressed, references to “wheat flour” as used hereinpreferably mean references to wheat flour per se as well as to wheatflour when present in a medium, such as a dough.

A preferred flour is wheat flour or rye flour or mixtures of wheat andrye flour. However, dough comprising flour derived from other types ofcereals such as for example from rice, maize, barley, and durra are alsocontemplated.

Preferably, the starch product is a bakery product.

More preferably, the starch product is a bread product.

Even more preferably, the starch product is a baked farinaceous breadproduct.

The term “baked farinaceous bread product” is understood to refer to anybaked product based on ground cereals and baked on a dough obtainable bymixing flour, water, and a leavening agent under dough formingconditions. It is, however, within the scope of the present inventionthat further components can be added to the dough mixture.

The term “amylase” is used in its normal sense—e.g. an enzyme that isinter alia capable of catalysing the degradation of starch. Inparticular they are hydrolases which are capable of cleaving α-D-(1→4)O-glycosidic linkages in starch.

The term “non-maltogenic exoamylase enzyme” means the enzyme does notinitially degrade starch to substantial amounts of maltose. In a highlypreferred aspect, the term also means the enzyme does not initiallydegrade starch to substantial amounts of maltose and glucose.

Before the present invention, non-maltogenic exoamylase enzymes had notbeen suggested for retarding the detrimental retrogradation of starchmedia, in particular starch gels.

A suitable assay for determining amylase activity in accordance with thepresent invention is presented later. For convenience, this assay iscalled the “Amylase Assay Protocol”.

Thus, preferably, the term “non-maltogenic exoamylase enzyme” means thatthe enzyme does not initially degrade starch to substantial amounts ofmaltose as analysed in accordance with the product determinationprocedure as described in the “Amylase Assay Protocol” presented herein.

In a preferred aspect, the non-maltogenic exoamylase can becharacterised in that if an amount of 0.7 units of said non-maltogenicexoamylase were to incubated for 15 minutes at a temperature of 50° C.at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maizestarch per ml buffered solution containing 50 mM 2-(N-morpholino)ethanesulfonic acid and 2 mM calcium chloride then the enzyme would yieldhydrolysis product(s) that would consist of one or more linearmalto-oligosaccharides of from two to ten D-glucopyranosyl units andoptionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis products would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

For ease of reference, and for the present purposes, the feature ofincubating an amount of 0.7 units of the non-maltogenic exoamylase for15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of an aqueoussolution of 10 mg preboiled waxy maize starch per ml buffered solutioncontaining 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calciumchloride, may be referred to as the “waxy maize starch incubation test”.

Thus, alternatively expressed, a preferred non-maltogenic exoamylase ischaracterised as having the ability in the waxy maize starch incubationtest to yield hydrolysis product(s) that would consist of one or morelinear malto-oligosaccharides of from two to ten D-glucopyranosyl unitsand optionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis product(s) would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

The hydrolysis products in the waxy maize starch incubation test includeone or more linear malto-oligosaccharides of from two to tenD-glucopyranosyl units and optionally glucose. The hydrolysis productsin the waxy maize starch incubation test may also include otherhydrolytic products. Nevertheless, the % weight amounts of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units arebased on the amount of the hydrolysis product that consists of one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and optionally glucose. In other words, the % weight amounts oflinear maltooligosaccharides of from three to ten D-glucopyranosyl unitsare not based on the amount of hydrolysis products other than one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and glucose.

The hydrolysis products can be analysed by any suitable means. Forexample, the hydrolysis products may be analysed by anion exchange HPLCusing a Dionex PA 100 column with pulsed amperometric detection andwith, for example, known linear maltooligosaccharides of from glucose tomaltoheptaose as standards.

For ease of reference, and for the pres nt purposes, the feature ofanalysing the hydrolysis product(s) using anion exchange HPLC using aDionex PA 100 column with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose used asstandards, can be referred to as “analysing by anion exchange”. Ofcourse, and as just indicated, other analytical techniques wouldsuffice, as well as other specific anion exchange techniques.

Thus, alternatively expressed, a preferred non-maltogenic exoamylase ischaracterised as having the ability in a waxy maize starch incubationtest to yield hydrolysis product(s) that would consist of one or morelinear malto-oligosaccharides of from two to ten D-glucopyranosyl unitsand optionally glucose, said hydrolysis products being capable of beinganalysed by anion exchange; such that at least 60%, preferably at least70%, more preferably at least 80% and most preferably at least 85% byweight of the said hydrolysis product(s) would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

As used herein with respect to the present invention, the term “linearmalto-oligosaccharide” is used in the normal sense as meaning 2-10 unitsof α-D-glucopyranose linked by an α-(1→4) bond.

The term “obtainable from P. saccharophila” means that the enzyme neednot necessarily be obtained from P. saccharophila. Instead, the enzymecould be prepared by use of recombinant DNA techniques.

The term “functional equivalent thereof” in relation to the enzyme beingobtainable from P. saccharophila means that the functional equivalentcould be obtained from other sources. The functionally equivalent enzymemay have a different amino acid sequence but will have non-maltogenicexoamylase activity. The functionally equivalent enzyme may have adifferent chemical structure and/or formula but will have non-maltogenicexoamylase activity. The functionally equivalent enzyme need notnecessarily have exactly the same non-maltogenic exoamylase activity asthe non-maltogenic exoamylase enzyme obtained from P. saccharophila. Forsome applications, preferably, the functionally equivalent enzyme has atleast the same activity profile as the enzyme obtained from P.saccharophila.

The term “obtainable from Bacililus clausii” means that the enzyme neednot necessarily be obtained from Bacillus clausii. Instead, the enzymecould be prepared by use of recombinant DNA techniques.

The term “functional equivalent thereof” in relation to the enzyme beingobtainable from Bacillus clausii means that the functional equivalentcould be obtained from other sources. The functionally equivalent enzymemay have a different amino acid sequence but will have non-maltogenicexoamylase activity. The functionally equivalent enzyme may have adifferent chemical structure and/or formula but will have non-maltogenicexoamylase activity. The functionally equivalent enzyme need notnecessarily have exactly the same non-maltogenic exoamylase activity asthe non-maltogenic exoamylase enzyme obtained from Bacillus clausii. Forsome applications, preferably, the functionally equivalent enzyme has atleast the same activity profile as the enzyme obtained from Bacillusclausii (such as the reactivity profile shown in FIG. 7).

General Comments

The present invention is based on the surprising finding thatnon-maltogenic exoamylases are highly effective in retarding or reducingdetrimental retrogradation, such as staling, in starch products, inparticular baked products.

We have found that non-maltogenic exoamylases according to the presentinvention can be more effective in retarding detrimental retrogradation,such as staling, in bread than the glucogenic and maltogenicexoamylases.

The reduction of detrimental retrogradation can be measured by standardtechniques known in the art. By way of example, some techniques arepresented later on in the section titled “Assay for the Measurement ofRetrogradation”.

In our studies, we have found that by incorporating a sufficient amountof activity of a non-maltogenic exoamylase, like for instance aexo-maltotetraohydrolase (EC 3.2.1.60), which has a sufficientthermostability, into a dough there is provided baked products withreduced, in some cases significantly reduced, detrimental retrogradationcompared to that of a control bread, such as under storage conditions.In contrast, the reducing effect on detrimental retrogradation ofincorporating the same amount of activity of a maltogenic exoamylasewith a comparable thermostability to that of the non-maltogenicexoamylase is significantly less. Thus, the anti-retrogradation effectof non-maltogenic exoamylase is more efficient than that of a maltogenicexoamylase. We believe that this difference may be, in part, due to theextent to which the amylopectin side chains are shortened. We alsobelieve that the anti-retrogradation effect may be even more pronouncedwhen using a non-maltogenic exoamylase according to the invention whichreleases maltoheptaose and/or maltooctaose and/or maltohexose.

In our studies we have also purified and characterised aproduct-specific amylase active at high pH producing maltohexaose. Thisamylase was isolated from an alkali-tolerant strain of Bacillus clausiiBT-21.

Furthermore, we have found that the retardation of detrimentalretrogradation that is obtainable by using non-maltogenic exoamylasesaccording to the present invention is dose responsive over a very widerange. This is in contrast to the effect from maltogenic exoamylases,which is rather limited and has a strongly decreasing dose response.

Amylases

In one aspect, the present invention provides the use of certainamylases to prepare starch products, such as bakery products. In thisrespect, the amylases—which are non-maltogenic exoamylases—retard orreduce the staling properties (i.e. lower the rate of staling) of thestarch product, in particular a baked farinaceous bread product.

Preferably, the amylase is in an isolated form and/or in a substantiallypure form. Here, the term “isolated” means that the enzyme is not in itsnatural environment.

As indicated above, the non-maltogenic exoamylase enzyme of the presentinvention does not initially degrade starch to substantial amounts ofmaltose.

According to the present invention, the non-maltogenic exoamylase iscapable of cleaving off linear maltooligosaccharides, predominantlyconsisting of from four to eight D-glucopyranosyl units, from thenon-reducing ends of the side chains of amylopectin. Non-maltogenicexoamylases having this characteristic and which are suitable for use inthe present invention are identified by their ability to hydrolysegelatinised waxy maize starch in the model system presented in theAmylase Assay Protocol (infra).

When incubated 15 min. under the described conditions in the AmylaseAssay Protocol, the non-maltogenic exoamylases which are suitable foruse according to the present invention would yield a hydrolysisproduct(s) that would consist of one or more linearmalto-oligosaccharides of from two to ten D-glucopyranosyl units andoptionally glucose, such that the product pattern of that hydrolysisproduct would consist of at least 60%, in particular at least 70%, morepreferably at least 80% and most preferably at least 90% by weight ofstarch hydrolysis degradation products other than maltose and glucose.

For a preferred aspect of the present invention, the non-maltogenicexoamylases which are suitable for use according to the presentinvention would provide when incubated 15 min. under the describedconditions for the waxy maize starch incubation test the said hydrolysisproduct, such that the hydrolysis product would have a product patternof at least 60%, in particular at least 70%, more preferably at least80% and most preferably at least 90% by weight of linearmalto-oligosaccharides of from three to ten D-glucopyranosyl units, inparticular linear maltooligosaccharides consisting of from four to eightD-glucopyranosyl units.

In a more preferred aspect of the present invention, the said hydrolysisproduct in said test would have a product pattern of at least 60%, inparticular at least 70%, more preferably at least 80% and mostpreferably at least 85% by weight of linear maltooligosaccharides of 4or 6 D-glucopyranosyl units.

In a more preferred aspect of the present invention, the said hydrolysisproduct in said test would have a product pattern of at least 60%, inparticular at least 70%, more preferably at least 80% and mostpreferably at least 85% by weight linear maltooligosaccharides of 4D-glucopyranosyl units.

In a more preferred aspect of the present invention, the said hydrolysisproduct in said test would have a product pattern of at least 60%, inparticular at least 70%, more preferably at least 80% and mostpreferably at least 85% by weight of linear maltooligosaccharides of 6D-glucopyranosyl units.

Preferentially, the non-maltogenic exoamylase does not substantiallyhydrolyze its primary products to convert them to glucose, maltose andmaltotriose. If that were the case, the primary products would competeas substrates with the amylopectin non-reducing chain ends for theenzyme, so that its anti-retrogradation efficiency would be reduced.

Thus, preferentially, the non-maltogenic exoamylase when incubated for300 min. under conditions similar to the waxy maize starch incubationtest but wherein the 15 min. period is extended to 300 min.—as an aside,and for convenience for the present purposes, this modified waxy maizestarch incubation test may be called the “extended waxy maize starchincubation test”—would still yield the said hydrolyis product whereinthe hydrolysis product would have a product pattern of at least 50%, inparticular at least 60%, more preferably at least 70% and mostpreferably at least 80% by weight of from four to eight D-glucopyranosylunits.

By way of example, a non-maltogenic exoamylase useful in the process ofthe present invention can be characterised in that it has the ability ina waxy maize starch incubation test to yield hydrolysis product(s) thatwould consist of one or more linear malto-oligosaccharides of from twoto ten D-glucopyranosyl units and optionally glucose; such that at least60%, preferably at least 70%, more preferably at least 80% and mostpreferably at least 85% by weight of the said hydrolysis product(s)would consist of linear maltooligosaccharides of from three to tenD-glucopyranosyl units, preferably of linear maltooligosaccharidesconsisting of from four to eight D-glucopyranosyl units; and wherein theenzyme is obtainable from P. saccharophila or is a functional equivalentthereof.

By way of further example, another non-maltogenic exoamylase useful inthe process of the present invention can be characterised in that it hasthe ability in a waxy maize starch incubation test to yield hydrolysisproduct(s) that would consist of one or more linearmalto-oligosaccharides of from two to ten D-glucopyranosyl units andoptionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis product(s) would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units; wherein the enzyme is obtainable fromBacillus clausii or is a functional equivalent thereof; and wherein theenzyme has a molecular weight of about 101,000 Da (as estimated bysodium dodecyl sulphate polyacrylamide electrophoresis) and/or theenzyme has an optimum of activity at pH 9.5 and 55° C.

Preferably, the non-maltogenic exoamylases which are suitable for useaccording to the present invention are active during baking andhydrolyse starch during and after the gelatinization of the starchgranules which starts at temperatures of about 55° C. The morethermostable the non-maltogenic exoamylase is the longer time it can beactive and thus the more antistaling effect it will provide. However,during baking above temperatures of about 85° C. the non-maltogenicexoamylase is preferentially gradually inactivated so that there issubstantially no activity after the baking process in the final bread.Therefore preferentially the non-maltogenic exoamylases suitable for useaccording to the present invention have an optimum temperature above 45°C. and below 98° C. when incubated for 15 min. at 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90° C. in a test tube with 4 ml of 10 mg/ml waxymaize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 prepared asdescribed above and assayed for release of hydrolysis products asdescribed above. Preferably the optimum temperature of thenon-maltogenic exoamylase is above 55° C. and below 95° C. and even morepreferably it is above 60° C. and below 90° C.

Non-maltogenic exoamylases which may be found to be less thermostablecan be improved by using protein engineering to become more thermostableand thus better suited for use according to present the invention. Thusthe use of non-maltogenic exoamylases modified to become morethermostable by protein engineering is encompassed by the presentinvention.

It is known that some non-maltogenic exoamylases can have some degree ofendoamylase activity. In some cases, this type of activity may need tobe reduced or eliminated since endoamylase activity can possiblynegatively effect the quality of the final bread product by producing asticky or gummy crumb due to the accumulation of branched dextrins.

Thus, in a preferred aspect, the non-maltogenic exoamylase of thepresent invention will have less than 0.5 endoamylase units (EAU) perunit of exoamylase activity.

Preferably the non-maltogenic exoamylases which are suitable for useaccording to the present invention have less than 0.05 EAU per unit ofexoamylase activity and more preferably less than 0.01 EAU per unit ofexoamylase activity.

The endoamylase units can be determined by use of the Endoamylase AssayProtocol presented below.

Examples of non-maltogenic exoamylases suitable for use according to thepresent invention include exo-maltotetraohydrolase (E.C.3.2.1.60),exo-maltopentaohydrolase and exo-maltohexaohydrolase (E.C.3.2.1.98)which hydrolyze 1,4-α-glucosidic linkages in amylaceous polysaccharidesso as to remove successive residues of maltotetraose, maltopentaose ormaltohexaose, respectively, from the non-reducing chain ends. Examplesare exo-maltotetraohydrolases of Pseudomonas saccharophila and P.stutzeri (EP-0 298 645 B1), exo-maltopentaohydrolases of an alkaliphilicGram-positive bacterium (U.S. Pat. No. 5,204,254) and of Pseudomonas sp.(Shida et al., Biosci. Biotechnol. Biochem., 1992, 56, 76-80) andexo-maltohexaohydrolases of Bacillus sp. #707 (Tsukamoto et al.,Biochem. Biophys. Res. Commun., 1988, 151, 25-31), B. circulans F2(Taniguchi, ACS Symp., 1991, Ser. 458, 111-124) and Aerobacter aerogenes(Kainuma et al., Biochim. Biophys. Acta, 1975, 410, 333-346).

Another example of a non-maltogenic exoamylase suitable for useaccording to the invention is the exoamylase from an alkalophilicBacillus strain, GM8901 (28). This is a non-maltogenic exoamylase whichproduces maltotetraose as well as maltopentaose and maltohexaose fromstarch.

Furthermore, non-maltogenic exoamylases suitable for use according tothe present invention also include exo-maltoheptaohydrolase orexo-maltooctaohydrolase which hydrolyze 1,4-α-glucosidic linkages inamylaceous polysaccharides so as to remove residues of maltoheptaose ormaltooctaose, respectively, from the non-reducing chain ends.Exo-maltoheptaohydrolase and exo-maltooctaohydrolase can be found eitherby screening wild type strains or can b developed from other amylolyticenzymes by protein engineering. Thus, non-maltogenic exoamylasesdeveloped by protein engineering from other amylolytic enzymes to becomenon-maltogenic exoamylases are also suitable for use in the presentinvention.

Novel Amylase

In one aspect, the present invention also provides a novel amylase thatis suitable for preparing starch products according to the presentinvention, such as bakery products. The novel amylase of the presentinvention is a non-maltogenic exoamylase. In our studies, we havechararacterised this new amylase that is suitable for the preparation offoodstuffs, in particular doughs for use in the preparation of bakeryproducts.

Thus, the present invention also provides a non-maltogenic exoamylase,wherein the non-maltogenic exoamylase is further characterised in thatit has the ability in a waxy maize starch incubation test to yieldhydrolysis product(s) that would consist of one or more linearmalto-oligosaccharides of from two to ten D-glucopyranosyl units andoptionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis product(s) would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units; wherein the enzyme is obtainable fromBacillus clausii or is a functional equivalent thereof; and wherein theenzyme has a molecular weight of about 101,000 Da (as estimated bysodium dodecyl sulphate polyacrylamide electrophoresis) and/or theenzyme has an optimum of activity at pH 9.5 and 55° C.

Preferably, the amylase is in an isolated form and/or in a substantiallypure form. Here, the term “isolated” means that the enzyme is not in itsnatural environment.

Antibodies

The enzymes of present invention can also be used to generateantibodies—such as by use of standard techniques. Thus, antibodies toeach enzyme according to the present invention may be raised. The oreach antibody can be used to screen for other suitable amylase enzymesaccording to the present invention. In addition, the or each antibodymay be used to isolate amounts of the enzyme of the present invention.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc. may be immunized by injection with theinhibitor or any portion, variant, homologue, fragment or derivativethereof or oligopeptide which retains immunogenic properties. Dependingon the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminium hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants which may be employed.

Monoclonal antibodies to the enzyme may be even prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosboret al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985)Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). Inaddition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,779) canbe adapted to produce inhibitor specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Improver Composition

As indicated, one aspect of the present invention relates to an improvercomposition for a starch product, in particular a dough and/or a bakedfarinaceous bread product made from the dough.

The improver composition comprises a non-maltogenic exoamylase accordingto the present invention and at least one further dough ingredient ordough additive.

According to the present invention the further dough ingredient or doughadditive can be any of the dough ingredients and dough additives whichare described above.

Expediently, the improver composition is a dry pulverulent compositioncomprising the non-maltogenic exoamylase according to the inventionadmixed with at least one further ingredient or additive. However, theimprover composition may also be a liquid preparation comprising thenon-maltogenic exoamylase according to the invention and at least onefurther ingredient or additive dissolved or dispersed in water or otherliquid. It will be understood that the amount of enzyme activity in theimprover composition will depend on the amounts and types of the furtheringredients and additives which form part of the improver composition.

Optionally, the improver composition may be in the form of a completemixture, a so-called pre-mixture, containing all of the dry ingredi ntsand additives for making a particular baked product.

Preparation of Starch Products

In accordance with one aspect of the present invention, the processcomprises forming the starch product by adding a suitable non-maltogenicexoamylase enzyme, such as one of the novel non-maltogenic exoamylaseenzymes presented herein, to a starch medium.

If the starch medium is a dough, then the dough is prepared by mixingtogether flour, water, the non-maltogenic exoamylase according to theinvention and other possible ingredients and additives.

By way of further example, if the starch product is a baked farinaceousbread product (which is a highly preferred embodiment), then the processcomprises mixing—in any suitable order—flour, water, and a leaveningagent under dough forming conditions and further adding a suitablenon-maltogenic exoamylase enzyme.

The leavening agent may be a chemical leavening agent such as sodiumbicarbonate or any strain of Saccharomyces cerevisiae (Baker's Yeast).

The non-maltogenic exoamylase can be added together with any doughingredient including the water or dough ingredient mixture or with anyadditive, or additive mixture.

The dough can be prepared by any conventional dough preparation methodcommon in the baking industry or in any other industry making flourdough based products.

Baking of farinaceous bread products such as for example white bread,bread made from bolted rye flour and wh at flour, rolls and the like istypically accomplished by baking the bread dough at oven temperatures inthe range of from 180 to 250° C. for about 15 to 60 minutes. During thebaking process a steep temperature gradient (200→120° C.) is prevailingin the outer dough layers where the characteristic crust of the bakedproduct is developed. However, owing to heat consumption due to steamgeneration, the temperature in the crumb is only close to 100° C. at theend of the baking process.

The non-maltogenic exoamylase can be added as a liquid preparation or asa dry pulverulent composition either comprising the enzyme as the soleactive component or in admixture with one or more additional doughingredient or dough additive.

In order to improve further the properties of the baked product andimpart distinctive qualities to the baked product further doughingredients and/or dough additives may be incorporated into the dough.Typically, such further added components may include dough ingredientssuch as salt, grains, fats and oils, sugar, dietary fibre substances,milk powder, gluten and dough additives such as emulsifiers, otherenzymes, hydrocolloids, flavouring agents, oxidising agents, mineralsand vitamins.

The emulsifiers are useful as dough strengtheners and crumb softeners.As dough strengtheners, the emulsifiers can provide tolerance withregard to resting time and tolerance to shock during the proofing.Furthermore, dough strengtheners will improve the tolerance of a givendough to variations in the fermentation time. Most dough strengthenersalso improve on the oven spring which means the increase in volume fromthe proofed to the baked goods. Lastly, dough strengtheners willemulsify any fats present in the recipe mixture.

The crumb softening, which is mainly a characteristic of themonoglycerides, is attributed to an interaction between the mulsifierand the amylose fraction of the starch leading to formation of insolubleinclusion complexes with the amylose which will not recrystallize uponcooling and which will not therefore contribute to firmness of the breadcrumb.

Suitable emulsifiers which may be used as further dough additivesinclude lecithin, polyoxyethylene stearat, mono- and diglycerides ofedible fatty acids, acetic acid esters of mono- and diglycerides ofedible fatty acids, lactic acid esters of mono- and diglycerides ofedible fatty acids, citric acid esters of mono- and diglycerides ofedible fatty acids, diacetyl tartaric acid esters of mono- anddiglycerides of edible fatty acids, sucrose esters of edible fattyacids, sodium stearoyl-2-lactylate, and calcium stearoyl-2-lactylate.

Other enzymes which are useful as further dough additives include asexamples oxidoreductases, such as glucose oxidase, hexose oxidase, andascorbate oxidase, hydrolases, such as lipases and esterases as well asglycosidases like α-amylase, pullulanase, and xylanase. Oxidoreductases,such as for example glucose oxidase and hexose oxidase, can be used fordough strengthening and control of volume of the baked products andxylanases and other hemicellulases may be added to improve doughhandling properties, crumb softness and bread volume. Lipases are usefulas dough strengtheners and crumb softeners and α-amylases and otheramylolytic enzymes may be incorporated into the dough to control breadvolume and further reduce crumb firmness.

The amount of the non-maltogenic exoamylase according to the presentinvention that is added is normally in an amount which results in thepresence in the finished dough of 50 to 100,000 units per kg of flour,preferably 100 to 50,000 units per kg of flour. In useful embodiments ofthe present invention, the amount is in the range of 200 to 20,000 unitsper kg of flour.

In the present context, 1 unit of the non-maltogenic exoamylase isdefined as the amount of enzyme which releases hydrolysis productsequivalent to 1 μmol of reducing sugar per min. when incubated at 50° C.in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2mM calcium chloride, pH 6.0 as described hereinafter.

Foodstuffs Prepared With Amylases

The present invention provides suitable amylases for use in themanufacture of a foodstuff. Typical foodstuffs, which also includeanimal feed, include dairy products, meat products, poultry products,fish products and bakery products.

Preferably, the foodstuff is a bakery product, such as the bakeryproducts described above. Typical bakery (baked) products incorporatedwithin the scope of the present invention include bread—such as loaves,rolls, buns, pizza bases etc.—pretzels, tortillas, cakes, cookies,biscuits, krackers etc.

Amylase Assay Protocol

The following system is used to characterize non-maltogenic exoamylaseswhich are suitable for use according to the present invention.

By way of initial background information, waxy maize amylopectin(obtainable as WAXILYS 200 from Roquette, France) is a starch with avery high amylopectin content (above 90%).

20 mg/ml of waxy maize starch is boiled for 3 min. in a buffer of 50 mMMES (2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0and subsequently incubated at 50° C. and used within half an hour.

One unit of the non-maltogenic exoamylase is defined as the amount ofenzyme which releases hydrolysis products equivalent to 1 μmol ofreducing sugar per min. when incubated at 50° C. in a test tube with 4ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH6.0 prepared as described above.

Reducing sugars are measured using maltose as standard and using thedinitrosalicylic acid method of Bernfeld, Methods Enzymol., (1954), 1,149-158 or another method known in the art for quantifying reducingsugars.

The hydrolysis product pattern of the non-maltogenic exoamylase isdetermined by incubating 0.7 units of non-maltogenic exoamylase for 15or 300 min. at 50° C. in a test tube with 4 ml of 10 mg/ml waxy maizestarch in the buffer prepared as described above. The reaction isstopped by immersing the test tube for 3 min. in a boiling water bath.

The hydrolysis products are analyzed and quantified by anion exchangeHPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxideand water as eluents, with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose asstandards. The response factor used for maltooctaose to maltodecaose isthe response factor found for maltoheptaose.

Endoamylase Assay Protocol

0.75 ml of enzyme solution is incubated with 6.75 ml of 0.5% (w/v) ofAZCL-amylose (azurine cross-linked amylose available from Megazyme,Ireland) in 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 2 mMcalcium chloride, pH 6.0 at 50° C. After 5, 10, 15, 20 and 25 minutes,respectively 1.0 ml of reaction mix is transferred to 4.0 ml of stopsolution consisting of 4% (w/v) TRIS (Tris(hydroxymethyl)aminomethane).

The stopped sample is filtered through a Whatman No. 1 filter and itsoptical density at 590 nm is measured against distilled water. Theenzyme solution assayed should be diluted so that the optical densityobtained is a linear function of time. The slope of the line for opticaldensity versus time is used to calculate the endoamylase activityrelative to the standard GRINDAMYL™ A1000 (available from DaniscoIngredients), which is defined to have 1000 endoamylase units (EAU) perg.

Assays for Measurement of Retrogradation (inc. Staling)

For evaluation of the antistaling effect of the non-maltogenicexoamylase of the present invention, the crumb firmness can be measured1, 3 and 7 days after baking by means of an Instron 4301 Universal FoodTexture Analyzer or similar equipment known in the art.

Another method used traditionally in the art and which is used toevaluate the effect on starch retrogradation of a non-maltogenicexoamylase according to the present invention is based on DSC(differential scanning calorimetry). Hereby the melting enthalpy ofretrograded amylopectin in bread crumb or crumb from a model systemdough baked with or without enzymes (control) is measured. The DSCequipment applied in the described examples is a Mettler-Toledo DSC 820run with a temperature gradient of 10° C. per min. from 20 to 95° C. Forpreparation of the samples 10-20 mg of crumb are weighed and transferredinto Mettler-Toledo aluminium pans which then are hermetically sealed.

The model system doughs used in the described examples contain standardwheat flour and optimal amounts of water or buffer with or without thenon-maltogenic exoamylase according to the present invention. They aremixed in a 10 or 50 g Brabender Farinograph for 6 or 7 min.,respectively. Samples of the doughs are placed in glass test tubes(15*0.8 cm) with a lid. These test tubes are subjected to a bakingprocess in a water bath starting with 30 min. incubation at 33° C.followed by heating from 33 to 95° C. with a gradient of 1.1° C. permin. and finally a 5 min. incubation at 95° C. Subsequently, the tubesare stored in a thermostat at 20° C. prior to DSC analysis.

Summary

In summary the present invention is based on the surprising finding thatnon-maltogenic exoamylases—which hydrolyse starch by cleaving off linearmaltooligosaccharides in the range of four to eight D-glucopyranosylunits from the non-reducing chain ends of amylopectin and whichpreferably have a sufficient degree of thermostability—are highlyeffective in retarding or reducing detrimental retrogradation in bakedproducts.

Deposits

The following sample was deposited in accordance with the BudapestTreaty at the recognised depositary DSMZ (Deutsche Sammlung vonMikrooganismen und Zellkulturen GmbH of Mascheroder Weg 1b, D-38124Braunschweig) on Mar. 12, 1999:

BT-21 DSM number DSM 12731

The present invention also encompasses sequences derivable and/orexpressable from those deposits and embodiments comprising the same, aswell as active fragments thereof.

INTRODUCTION TO THE EXAMPLES SECTION AND THE FIGURES

The present invention will now be described, by way of example only,with reference to the accompanying drawings in which:

FIG. 1 shows a graph;

FIG. 2 shows a graph;

FIG. 3 shows a graph;

FIG. 4 shows a graph;

FIG. 5 shows a graph;

FIG. 6 shows a trace; and

FIG. 7 shows a graph.

In more detail:

FIG. 1. Extracellular amylolytic activity (mU/mL) in liquid cultures ofB. clausii BT-21 cultured in 2% starch substrates at 45° C. ♦ solublestarch, ● amylopectin, ♦ corn starch, ▪ whole brown rice. Bars indicatethe standard deviation.

FIG. 2. Effect of pH on the activity of the product-specific amylase.Effect of pH at 55° C.

FIG. 3. Effect of temperature on the activity of the product-specificamylase. Effect of temperature at pH 9.5 ▪ with 5 mM CaCl₂ □ withoutCaCl₂.

FIG. 4. Thermostability tested as residual activity of theproduct-specific amylase after incubation at increasing temperatures atpH 9.5 with 5 mM CaCl₂.

FIG. 5. Products (in mM) formed by incubating the product-specificamylase (505 mU/mL) with 1% soluble starch and 5 mM CaCl₂ at 55° C. andpH 9.5. ◯ glucose, x maltose, □ maltotriose, ▴ maltotetraose, ●maltopentaose, ▪ maltohexaose.

FIG. 6. HPAEC-PAD trace obtained by incubating the product-specificamylase (505 mU/ mL) with 1% soluble starch at pH 9.5 and 55° C. A)Soluble starch without enzyme, B) Incubation with enzyme for 30 min.

FIG. 7. Determination of endo-and exo-activity of B. clausii BT-21product-specific amylase compared to amylases of known starch cleavageaction. The blue colour formation (% of maximum) is plotted against theproduction of mM maltose. The slope of the curves indicate theprevalence of endo- or exo-activity

EXAMPLE Section A Example 1 Fermentation and Production of Pseudomonassaccharophila Non-maltogenic Exoamylase

1.1 Production

P. saccharophila strain IAM No. 1544 was obtained from the IAM CultureCollection, Inst. of Molecular and Cellular Biosciences, University ofTokyo, Japan.

Fermentation of the strain was performed in an Applikon ADI 3 literbioreactor with 2 liter of working volume and under the followingconditions:

Temperature: 30° C. Stirring rate: 1000 rpm Aeration: 1 volume air pervolume medium per minute pH: constant pH 7.4 by adjustment with 2Msodium hydroxide and 10% (w/v) hydrochloric acid Medium: Bacto Tryptone20 g/l Bacto Yeast extract 20 g/l Starch 20 g/l Na₂HPO₄·2H₂O 5.6 g/l KH₂PO₄ 1.5 g/l 

After 1 day of fermentation the fermentation broth was centrifuged andfiltered to remove the cells. The activity of non-maltogenic exoamylasein the cell free broth was 5 units per ml determined as described above.

1.2 Purification of P. saccharophila Non-maltogenic Exoamylase

P. saccharophila non-maltogenic exoamylase was partially purified byhydrophobic interaction chromatography using a 150 ml Phenyl SepharoseFF low sub column (Pharmacia, Sweden) equilibrated with A-buffer being200 mM sodium sulfate, 50 mM triethanolamine, 2 mM calcium chloride, pH7.2. Filtered fermentation broth (500 ml) was adjusted to 200 mM sodiumsulfate and pH 7.2 and loaded onto the column. The non-maltogenicexoamylase was eluted with a linearly decreasing gradient of sodiumsulfate in A-buffer. The fractions containing exoamylase activity werepooled.

The pooled fractions were diluted three times with water and furtherpurified by anion-exchange chromatography on a 150 ml Q-Sepharose FF(Pharmacia) column equilibrated with A-buffer being 50 mMtriethanolamine, 5 mM calcium chloride, pH 7.5. The non-maltogenicexoamylase was eluted with a linear gradient of 0 to 1 M sodium chloridein A-buffer. The fractions containing exoamylase activity were pooled.This partially purified preparation was used for the tests describedbelow. It had an activity of 14.7 units per ml and only one band ofamylase activity when tested in a polyacrylamide gel electrophoresissystem stained for amylase activity.

1.3 Characterization of P. saccharophila Non-maltogenic Exoamylase

By way of introduction, the DNA sequence for the gene encoding P.saccharophila exo-amylase (which we call PS4) has been published by Zhouet al (Zhou J H, Baba T, Takano T, Kobayashi S, Arai Y (1989) FEBS Lett1989 Sep. 11,; 255(1):37-41 “Nucleotide sequence of themaltotetraohydrolase gene from Pseudomonas saccharophila.”. In addition,the DNA sequence can be accessed in GenBank with accession numberX16732.

We have now determined the MW of the purified PS4 enzyme by massspectrometry (MALDI-TOFF) to be 57500±500 D which is in accordance withthe theoretical MW of 57741 D derived from the sequence.

The optimum temperature and pH of PS4 are 45° C. and pH 6.5 according toZhou et al (Zhou J H, Baba T, Takano T, Kobayashi S, Arai Y (1992)Carbohydr Res 1992 January; 223:255-61 “Properties of the enzymeexpressed by the Pseudomonas saccharophila maltotetraohydrolase gene(mta) in Escherichia coli.”)

The hydrolysis pattern of the non-maltogenic exoamylase was determinedby analyzing the hydrolysis products generated by incubating 0.7 unitsof partially purified non-maltogenic exoamylase for 15 or 300 min. at50° C. in a test tube with 4 ml of 10 mg/ml waxy maize starch asdescribed above.

The patterns of hydrolysis products detected after 15 min. and 300 min.are shown in Table 1 and indicate that P. saccharophila produces anon-maltogenic exoamylase as defined in the present invention and thatthis enzyme releases maltotetraose as the predominant product accountingfor 85.8 wt % and 93.0 wt % after 15 and 300 min. hydrolysis,respectively.

TABLE 1 Hydrolysis products of glucose to maltodecaose of P.saccharophila non- maltogenic exoamylase Time DP 1 2 3 4 5 6 7 8 9 10Total 15 min. μg/ml 1 13 6 1609 13 29 40 70 51 43 1875 15 min. % 0.0 0.70.3 85.8 0.7 1.5 2.1 3.7 2.7 2.3 99.8 300 min. μg/ml 14 149 135 5354 816 4 12 0 0 5756 300 min. % 0.3 2.6 2.3 93.0 1.4 0.1 0.1 0.2 0.0 0.0100.0

Example 2 Baking Test of P. saccharophila Non-maltogenic Exoamylase

A baking test was set up to test the antifirming effect of P.saccharophila non-maltogenic exoamylase. A recipe for Danish Toast Breadwas used. It contains flour (2000 g), dry yeast (30 g), sugar (30 g),salt (30 g) and water (approximately 1200 g corresponding to a doughconsistency of 400 Brabender Units (BU)+60 g of additional water tocompensate for the dry yeast used) are mixed in a Hobart mixer (modelA-200) for 2 minutes at slow speed and for 12 minutes at high speed. Thedough temperature is 26° C. at the end of mixing. The dough is restedfor 10 minutes at 30° C. after which the dough is divided in doughpieces of 750 g. The dough pieces rest for 5 minutes in a proofingcabinet at a temperature of 33° C. and a relative humidity of 85%. Thedough pieces are then moulded on Glimek moulder (type LR-67) with thefollowing settings 1:4, 2:2, 3:14 and 4:12, after which the mouldeddough pieces are transferred to baking tins and proofed in a proofingcabinet for 50 minutes at a temperature of 33° C. and a relativehumidity of 85%. Finally, the proofed dough pieces are baked for 40minutes at a temperature of 220° C., with 10 seconds steam, in a Wachteloven (model AE 416/38 COM).

A partially purified preparation of P. saccharophila non-maltogenicexoamylase was added to the dough dosed at 1470 units per kg of flour.After baking the breads with or without the non-maltogenic exoamylasewere cooled to 20° C. and thereafter stored at 20° C. in plastic bags.Firmness was determined by means of an Instron 4301 Universal FoodTexture Analyzer on day 3 and day 7 after baking as the mean of 10slices of one bread for day 3 and the mean of 2 breads with 10 slicesper bread measured for day. Table 2 shows that a lower firmness in thebreads with enzyme added was observed for both days.

TABLE 2 Antifirming effect of P. saccharophila non-maltogenic exoamylaseTreatment Firmness day 3 Firmness day 7 Control 49 71 PS4 43 59

Table 3 shows that for day 7 the antifirming effect of the P.saccharophila non-maltogenic exoamylase is statistically significant onthe 95% confidence level.

TABLE 3 Statistical analysis of the antifirming effect of P.saccharophila non- maltogenic exoamylase ANOVA Table for Firmness day 7by Enzyme Analysis of Variance Source Sum of Squares Df Mean SquareF-Ratio P-Value Between groups 144,0 1 144,0 72,00 0,0136 Within groups 4,0 2  2,0 Total (Corr.) 148,0 3 The StatAdvisor The ANOVA tabledecomposes the variance of Firmness day 7 into two components: abetween-group component and a within-group component. The F-ratio, whichin this case equals 72,0, is a ratio of the between-group estimate tothe within-group estimate. Since the P-value of the F-test is less than0,05, there is a statistically significant difference between the meanFirmness day 7 from one level of Enzyme to another at the 95,0%confidence level. To determine which means are #significantly differentfrom which others, select Multiple Range Tests from the list of TabularOptions.

Example 3

The following describes our cloning and expression of the mta geneencoding non-maltogenic exoamylase from Pseudomonas saccharophila inEscherichia coli MC1061.

In this respect, P. saccharophila IAM 1520 was grown in 2 ml LB mediumand cells were harvested by centrifugation 10 min 20.000×g. Total DNAwas isolated using a slightly modified miniprep protocol. The cells wereresuspended in 300 μl resuspension buffer (50 mM Tris-HCl, pH 8.0; 10 mMEDTA; 100 μg/ml RNase A) after which the cells were disrupted using aFastprep FP120 (BIO101; California). Following disruption, 300 μl lysisbuffer (200 mM NaOH; 1% SDS) and 300 μl neutralization buffer (3.0 Mpotassium acetate, pH 5.5) were added. After centrifugation at 20,000×gfor 15 min at 4° C., the supernatant was collected and 0.6 volumesisopropanol was added. The DNA was precipitated by centrifugation at20,000×g for 30 min at 4° C., washed with 70% ethanol, and redissolvedin 100 μl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

For PCR amplification 4 different PCR primers were designed:

#1 ATG ACG AGG TCC TTG TTT TTC (SEQ ID NO: 1) pos  213-233 #2 GCT CCTGAT ACG ACA GCG (SEQ ID NO: 2) pos 2403 -2386 #3 GCC ATG GAT CAG GCC GGCAAG AGC CCG (SEQ ID NO: 3) pos  663-683 #4 TGG ATC CTC AGA ACG AGC CGCTGG T (SEQ ID NO: 4) pos 2258-2238

The positions refer to the sequence for mta found in GenBank accessionnumber X16732. The primers with the higher number first are antisenseprimers and the sequences are the complementary sequences. In bold arerepresented nucleotides which are not complementary to the template DNA,and underlined are introduced restriction sites.) Primer #3 introduces aunique Nco1 site, and primer #4 a BamHI site which are used for thefollowing cloning in the expression vector pBAD/gIII (Invitrogen).

A first PCR amplification using the following combination of primers wasperformed:#1+#2 giving a fragment on 2190 bp  reaction 1with 50-150 ng genomic IAM1520 DNA as template, using the Expand DNApolymerase (Boehringer Mannheim; Germany) according to the instructionsof the manufacturer and the following amplification protocol:

94° C. 2 min, (94° C. 1 min, 58° C 2min , 72° C. 2 min) for 35 cyclesand finally 72° C. to 5 min.

A 2190 bp fragment was isolated from gel using the ‘gene clean kit’(BIO101; California). The fragment was used as template DNA in a secondPCR with the following primer combination:#3+#4 giving a fragment on 1605 bp  reaction 2using the same amplification protocol as described above.

A 1605 bp fragment was purified and cloned into pCR-BLUNT vector(Invitrogen) according to the instructions of the manufacturer. Thesequence of the cloned fragment was confirmed by sequencing using thesingle dye sequencing technology and a ALF sequencer (Pharmacia; Sweden)using the universal and reverse primers, and four labeled internalprimers.

CAT CGT AGA GCA CCT CCA (SEQ ID NO: 5)  999-982 GAT CAT CAA GGA CTG GTCC (SEQ ID NO: 6) 1382-1400 CTT GAG AGC GAA GTC GAA C (SEQ ID NO: 7)1439-1421 GAC TTC ATC CGC CAG CTG AT (SEQ ID NO: 8) 1689-1708

The positions refer to the sequence for mta found in GenBank accessionnumber X16732. The primers with the higher number first are antisenseprimers and the sequences are the complementary sequences.

After confirming the sequence, the mta gene was cloned into theexpression vector pBAD/gIII (Invitrogen). The mta gene was released frompCR-BLUNT by digestion with BamHI followed by blunting with Klenowfragment and digestion with NcoI, and a 1602 bp fragment was purified.The expression vector pBAD/gIII was digested with NcoI and PmeI andpurified. After ligation the obtained expression construct wastransformed into Escherichia coli MC1061 cells, and the protein wasexpressed according to the pBAD/gIII manual (Invitrogen).

Example 4 Comparison of the Effect of a Non-maltogenic and a MaltogenicExoamylase on Starch Retrogradation

Sweet potato β-amylase (EC 3.2.1.2; obtainable from Sigma with productno. A7005) is a maltogenic exoamylase releasing maltose from thenon-reducing ends of starch. The thermostability of this maltogenicexoamylase is similar to that of P. saccharophila non-maltogenicexoamylase as indicated by the residual activities after incubation for15 minutes at temperatures from 45 to 75° C. in 50 mM sodium citrate, 5mM calcium chloride, pH 6.5 (Table 4).

TABLE 4 Residual activities of sweet potato β-amylase and P.saccharophila non-maltogenic exoamylase after incubation at increasingtemperatures (in %)^(a) Incubation temperature (° C.) 45 50 55 60 65 7075 Sweet potato β-amylase activity 100 117 55 12 5 4 4 (%) P.saccharophila exoamylase 100 68 29 10 7 6 6 activity (%) ^(a)Activityafter incubation at 45° C. set to 100%.

The effects of both enzymes on starch retrogradation have been tested byDSC analysis of the baked and stored products from model system doughsas described in “Assays for measurement of retrogradation and staling”.For this test 485 units of P. saccharophila non-maltogenic exoamylaseand 735 units of sweet potato β-amylase assayed according to “Amylaseassay protocol” were used in the doughs. The doughs were prepared of 50g of standard Danish wheat flour (Danisco 98022) with 30.7 ml 50 mMsodium citrate, 5 mM calcium chloride, pH 6.5 without (control) or withenzymes added.

After 7 days of storage the amount of retrograded amylopectin wasquantified by measuring its melting enthalpy. By statistical analysis itwas found that both enzymes significantly reduce starch retrogradation(Table 5); i.e. P. saccharophila non-maltogenic exoamylase reduces theamount of retrograded amylopectin on day 7 to 86% (1.77 J/g) whereassweet potato β-amylase lowers it to 96% (1.96 J/g) of the control (2.05J/g). In conclusion, P. saccharophila non-maltogenic exoamylase isclearly much more efficient for reducing retrogradation and staling thanthe maltogenic amylase with a comparable thermostability.

TABLE 5 Effect of P. saccharophila non-maltogenic exoamylase (PS4) andsweet potato β-amylase (SP2) on starch retrogradation based on measuringthe melting enthalpy of retrograded amylopectin (in J/g) 7 days afterbaking Multiple Range Tests for Enthalpy by Treatment Method: 95,0percent LSD Treatment Count Mean PS4 13 1,77154 SP2 22 1,96273 Control 6 2,05167 Contrast Difference +/− Limits Control - PS4 *0,280128 0,0941314 Control - SP2 *0,0889394 0,087841 PS4 - SP2 *−0,191189 0,06672*denotes a statistically significant difference. This table applies amultiple comparison procedure to determine which means are significantlydifferent from which others. The bottom half of the output shows theestimated difference between each pair of means. An asterisk has beenplaced next to 3 pairs, indicating that these pairs show statisticallysignificant differences at the 95,0% confidence level. The methodcurrently being used to discriminate among the means is Fisher's leastsignificant difference (LSD) procedure.

EXAMPLE Section B Materials and M thods

Materials.—Amylopectin and amylose from corn, corn starch,carboxymethylcellulose (CMC), bovine serum albumine (BSA), dextran,pullulan, maltose, maltotriose, and a mixture of maltotetraose tomaltodecaose were obtained from Sigma Chemical Co., St. Louis, U.S.A.Soluble starch was obtained from Merck KGaA, Darmstadt, Germany. Yeastextract and tryptone were obtained from Difco Laboratories, Detroit,USA. Whole brown rice from Neue Aligemeine Reisgesellschaft mbH,Hamburg, Germany was used. Pharmaceutical grade α-, β-, andγ-cyclodextrin were obtained from Wacker Chemie Danmark Aps, Glostrup,Denmark. Maltotetraose was prepared as described previously [32]. Allchemicals were, unless stated otherwise, of analytical grade.

Isolation of B. clausii BT-21.—The strain was isolated from a soilsample collected in Assens, Denmark, identified by DSMZ (DeutscheSammlung von Mikroorganismen und Zelikulturen GmbH, Braunschweig,Germany)

Production of the enzyme.—B. clausii BT-21 was grown in an optimisedliquid medium composed of 2.0% soluble starch from potato, amylopectinfrom corn, corn starch, or whole brown rice, 0.5% yeast extract, 0.5%tryptone, 0.1% KH₂PO₄, 0.1%, Na₂HPO₄, 0.02% MgSO₄.7H₂O, 0.02%CaCl₂.2H₂O, and 0.1% (NH₄)₂ SO₄. After autoclaving, a sterile Na₂CO₃solution was added to a final concentration of 1% (approximately pH 10).A 1 mL spore suspension in glycerol (stored at −80° C.) was used toinoculate 100 ml of the actual medium and incubated at 45° C. for 18 hin a shaking incubator (New Brunswick Scientific, Edison, N. J., U.S.A.)at 200 rpm. Two mL of this culture was used to inoculate a shake flaskwith 200 mL medium and incubated at 45° C. in a shaking incubator.Aliquots were taken in regular intervals and the OD at 600 nm wasmeasured to determine the growth of the strain in the media. Samples (4mL) were centrifuged at 9600 rpm for 10 min at 4° C. and the pH andamylase activity was determined. All growth experiments were carried outin triplicate. The mean value (X=(Σ^(n) _(i=1)X_(i))/n) and the standarddeviation values (std.=√(Σ^(n) _(i=1)(X_(i)−X)/(n−1)) were determined.

Purification of the product-specific amylase.—After growth of B. clausiiBT-21 on whole brown rice for 52 h, the cells and the whole rice grainswere removed from the extracellular fluid (1000 mL) by centrifugation at9600 rpm for 15 min at 4° C. The product-specific amylase was purifiedusing an affinity gel prepared by covalently binding β-cyclodextrin toan epoxy-activated sepharose 6B matrix (Pharmacia Biotech, Uppsala,Sweden) [33]. The extracellular cell-free supernatant was incubated with12 g of gel while shaking for 1 h at 4° C. The supernatant was thenremoved by centrifugation at 9600 rpm for 10 min at 4° C. Unboundprotein was removed by washing the gel with 75 mL 50 mM phosphate bufferpH 8.0 followed by centrifugation. The washing step was repeated 7times. Bound protein was eluted with 45 mL of 50 mM phosphate buffer pH8.0 containing 10 mM α-cyclodextrin followed by centrifugation. Theelution step was repeated 4 times. α-Cyclodextrin was used for elutionof the enzyme, since β-and γ-cyclodextrin interfered with the proteindetermination method of Bradford (1976) [34]. The α-cyclodextrin wasthen removed by dialysis (6-8 kDa Spectra/Por dialysis membrane, TheSpectrum Companies, Gardena, California, U.S.A.) against 5 L 10 mMtriethanolamin pH 7.5 while stirring at 4° C. The buffer was changedafter 2 h followed by an additional 12 h of dialysis. The dialysis bagswere placed in CMC to concentrate the sample. Ten mL were applied to aHiTrap Q column (5 mL prepacked, Pharmacia Biotech, Uppsala, Sweden)using a FPLC-system (Pharmacia, Uppsala, Sweden). The proteins wereeluted at the rate of 1.0 mL/min with 25 mL 10 mM triethanolamin pH 7.5followed by a gradient of 20 mM NaCl/min in 10 mM triethanolamin pH 7.5.The enzyme was eluted at 0.5 M NaCl. The protein content was estimatedby the method of Bradford, (1976) [34] using the BIO-RAD Protein Assay(Bio-Rad Laboratories, Hercules, Calif., USA). BSA was used as standard.

Gel electrophoresis.—15 μL samples were analysed by native tris-glycinegel, 10%, as described by [35]. The gel was then placed in 50 mMphosphate buffer at pH 6.5 and shaken for 30 min. A 1% (w/v) solublestarch solution was incubated with the gel while shaking for 45 minutes.After washing in buffer solution, the gel was incubated with an iodinesolution (4 mM I2, 160 mM KI) and decoloured with buffer. Destainedbands indicated starch hydrolysis activity.

SDS-PAGE (10%) was performed according to [36] followed by silverstaining [37]. A SDS-PAGE broad range molecular weight standard (Bio-Radlaboratories, Hercules, Calif., U.S.A.) was used.

Enzyme assay.—Two ml soluble starch solution (1.25%) in 0.1 M boratebuffer pH 10.0 was incubated with 0.5 mL enzyme solution for 2 h at 45°C. The reaction was stopped by boiling the mixture for 10 min. Theformation of reducing sugars was determined with the CuSO₄/bicinchonateassay [38] and calculated as mM maltose equivalent formed. One unit ofactivity corresponded to the amount of enzyme that produced 1 μmolmaltose equivalent/min at pH 10.0 and 45° C.

Enzyme characterisation.—For the determination of the temperatureoptimum, the purified enzyme was incubated in a final concentration of1% soluble starch in 0.1 M borate buffer pH 10.0 (with or without theaddition of 5 mM CaCl₂) for 15 min at temperatures from 30° C. to 90° C.Determination of the temperature stability was performed by incubationof the purified enzyme in 50 mM glycine-NaOH buffer pH 9.5 containing 5mM CaCl₂ for 30 min at 30, 40, 50, 55, 60, 70, 80, and 90° C. Residualactivity was determined by incubation of the heat-treated enzyme in afinal concentration of 1% soluble starch in 50 mM glycine-NaOH buffer pH9.5 at 55° C. for 15 min. The pH optimum was determined by incubation ofthe purified enzyme in a final concentration of 1% soluble starch indifferent buffers at 55° C. for 15 min. The buffers used were 50 mMcitrate (pH 4.0 to 6.0), 50 mM tris-maleate (pH 6.5 to 8.5), and 50 mMglycine-NaOH (pH 9.0 to 11.0).

An I₂-Kl solution (0.02% 12 and 0.2% KI) was prepared according to Fuwa,1954 [39]. The starch-iodine blue colour formation was measured induplicates with the following modifications. A sample of 500 μL waswithdrawn from the enzymatic hydrolysis of soluble starch at differenttime intervals. Then 250 μL HCl and 250 μL I₂-KI-solution were added andmixed. Deionised water (4.0 mL) was added and mixing was repeated. Theformation of a blue colour was measured spectrophotometrically at 600nm.

The hydrolysis of different substrates by the purified enzyme was testedwith soluble starch from potato, amylopectin from corn, dextran,pullulan (1%), amylose (0.1%), and 10 mM α-, β-, and γ-cyclodextrin. Thesubstrates were dissolved in 50 mM glycine-NaOH buffer with 5 mM CaCl₂at pH 9.5 and the purified enzyme was added (505 mU/mL). The varioussubstrates were incubated at 55° C. and samples were withdrawn atdifferent time intervals. The reaction was stopped by boiling for 10 minand the samples were analysed as described below.

The hydrolysis of malto-oligosaccharides by the purified enzyme wastested with maltose, maltotriose, and maltotetraose in a finalconcentration of 2 mM and a mixture of maltotetraose to maltodecaose (5mM). The malto-oligosaccharides were dissolved in 50 mM borate bufferwith 5 mM CaCl₂ at pH 9.5 and the purified enzyme was added (147 mU/mL).The substrates were incubated at 55° C. and samples were withdrawn atdifferent time intervals. The reaction was stopped by boiling for 10 minand the samples were analysed as described below.

Analysis of hydrolysis products.—Hydrolysis products were detected usinghigh performance anion exchange chromatography with pulsed amperometricdetection (HPAEC-PAD). A CarboPac PA-1 column (Dionex Corporation,Sunnyvale, Calif., U.S.A) was used with a gradient of 1.0 M Na-acetatefrom 0 to 60% over 30 min in 100 mM NaOH and a flow rate of 1.0 mL/minon a Dionex DX-300 or DX-500 system. Starch hydrolysis products wereidentified by comparison of their retention times with glucose, maltose,maltotriose, maltotetraose, maltopentaose, and maltohexaose. Since theretention times of homologous linear malto-oligosaccharides increaseswith the degree of polymerisation, linear malto-oligosaccharides ofintermediate DP could be easily identified [40, 41].

Results and Discussion

Identification of B. clausii BT-21.—According to its fatty acidcomposition, the strain showed similarity to the genus Bacillus. Apartial sequencing of the 16SrDNA showed a similarity of 99.4% to B.clausii. The physiological properties of the alkali-tolerant strainconfirmed this identification.

Production of amylase activity by B. clausii BT-21.—Soluble starch frompotato, corn starch, amylopectin from corn and whole brown rice resultedin different levels of extracellular amylase activity in the medium.While, corn starch contain more lipids and no phosphorus compared topotato starch amylopectin from corn has a highly branched structurecontaining α-D-(1→6) O-glycosidic linkages. These three types of starchare accessible for enzymes after heat gelatinisation, while whole brownrice contains a less accessible starch encapsulated in the rice grains.The amylase activities in the extracellular fluid of liquid cultureswith the different starch substrates are shown in FIG. 1. The highestamylolytic activity was obtained with whole brown rice as a substrate.This indicates that the presence of a less accessible starch substrateresulted in an increased production of extracellular amylolyticactivities by B. clausii BT-21. Similar results were obtained with wheatbran, which was however difficult to remove from the extracellular fluidprior to purification of the enzyme. Carbon sources such as galactose,glycogen, and inulin have previously been reported as suitable foramylase production by B. licheniformis [27] and soluble starch has beenfound as the best substrate for the production of an amylase by B.stearothermophilus [28 ]. However, none of these studies has included aless accessible starch substrate.

Purification of the product-specific amylase.—The enzyme was purified byaffinity chromatography with β-CD Sepharose 6B followed byanion-exchange chromatography (Table 6).

TABLE 6 Purification of the product-specific amylase from B. clausiiBT-21. Total Total Specific Purifi- Volume Activity activity proteinactivity (mU/ % cation (mL) (mU/mL) (mU) (mg) mg protein) recoveryfactor Extracellular 4000 155 620,000  424   731 100 1 fluid β-CDaffinity 704.5 88 62,137 8.5 3,647 10.0 5 chromatography Concentration172.6 254 43,840 4.8 4,581 7.1 6.3 and dialysis Anion-exchange 215 25154,051 2.0 13,493  8.7 18.5 chromatography

Activity stained native PAGE indicated the presence of 3 amylolyticactivities in the extracellular fluid. The product-specific enzyme wascompletely separated from the other amylolytic activities after β-CDaffinity chromatography followed by anion-exchange chromatography.SDS-PAGE of the purified enzyme preparation indicated that theproduct-specific amylase has been purified to homogeneity and has anapparent molecular weight of approximately 101 kDa. Cyclodextrinsepharose 6B affinity chromatography has been previously used for thepurification of an α-amylase as a final purification step after removalof other amylases by anion-exchange chromatography [29]. The enzymerecovery of 8.7% and the purification factor of 18.5 obtained for theproduct-specific amylase were similar to the values reported by theseauthors.

Characterisation of the product-specific amylase.—The purified enzymeshowed an optimum of activity at pH 9.5 (FIG. 2) while optimumtemperature for its activity was at 55° C. with or without the presenceof 5 mM CaCl₂ (FIG. 3). The thermostability of the enzyme at pH 9.5 inthe presence of 5 mM CaCl₂ is shown in FIG. 4. At temperatures above 55°C., the enzyme lost 75% of its maximum activity during a 30 minincubation period. Five other product-specific amylases formingmaltohexaose [23], maltopentaose [24], and maltotetraose [26] also showan alkaline pH optimum. The molecular weight of these enzymes wereestimated to be 59, 73 and 80 kDa [23], 180 kDa [24], and 97 kDa [26]while the product-specific amylase from B. clausii BT-21 showed anestimated molecular weight of 101 kDa. Most of the product-specificamylases and α-amylases show a lower molecular weight in the range of50-65 kDa [3, 16, 17, 19, and 21]. The temperature optimum of about 55°C. was similar to the ones reported for the above product-specificamylases [23, 24, and 26].

The purified product-specific amylase hydrolysed soluble starch after 1h of incubation mainly to maltohexaose and maltopentaose (52% and 19% oftotal hydrolysed products) as the main initial products of low DP (FIG.5). After 2 h of incubation, the amount of maltohexaose and after 4 hthe amount of maltopentaose decreased while the amounts ofmaltotetraose, maltotriose, maltose and glucose increased. Theseproducts accumulated after prolonged hydrolysis indicating that theywere not further hydrolysed. After 24 h of starch hydrolysis, theamounts of malto-oligosaccharides was (3%) maltohexaose, (4%)maltopentaose, (41%) maltotetraose, (13%) maltotriose, (16%) maltose and(4%) glucose. The high performance anion-exchange chromatography withpulsed amperometric detection (HPAEC-PAD) trace obtained after 30 minstarch hydrolysis (FIG. 6) shows that starch hydrolysis products largerthan maltohexaose (DP6) were absent. A time course study of thehydrolysis of soluble starch by a maltohexaose-forming product-specificamylase has also shown that maltohexaose was produced preferentially inthe early stage of hydrolysis [23]. Kim et al (1995) [26] found that theinitial hydrolysis product after 1 h hydrolysis of starch with amaltotetraose-forming product-specific amylase was mainly maltohexaose(54%) followed by a gradual increase in the amounts of maltotetraose andmaltose while the amount of maltohexaose decreased. After 20 h, thecomposition of malto-oligosaccharides had changed to 0.6% maltohexaose,1.3% maltopentaose, 53.2% maltotetraose, 8.3% maltotriose, 27.6% maltoseand 9% glucose.

To examine further the mode of action of the enzyme during starchhydrolysis, different substrates were incubated with the purified enzyme(Table 7).

TABLE 7 Malto-oligosaccharides in the range DP1 to DP6 formed by thehydrolysis of various starch substrates by the purified product-specificenzyme (67 mU/mL). Data are indicated as wt % glucose formed compared tothe initial amount of substrate Hydrolysis Product formed (%) Substratetime (min) DP1 DP2 DP3 DP4 DP5 DP6 Soluble starch 15 0 0 0 <0.1 0.9 7.2(1%) 240 0 0 0 0.6 7.0 17.8 Amylose (0.1%) 15 0.2 0 0 0 2.4 13.6 240 1.50 4.1 7.7 6.3 20.0 Amylopectin 15 <0.1 0 0 0 0.8 3.6 (1%) 240 <0.1 0.50.1 0.9 9.2 24.8

Starches are composed of amylose (20-30%) and amylopectin (80-70%).Amylose is an α-D-(1→4) O-glycosidically linked linear glucan, whileamylopectin is a branched glucan due to the presence of α-D-(1→6)O-glycosidic linkages in the molecule. The product-specific amylase mostreadily hydrolysed amylopectin indicated by the formation ofmaltopentaose (9.2%) and maltohexaose (24.8%) compared to soluble starch(7% and 17.8%) and amylose (6.3% and 20%). The enzyme did not hydrolysepullulan, an α-(1→6) O-glycosidic linked glucan composed of amaltotriose backbone, or dextran, an α-(1→6) O-glycosidically linkedglucan with branches attached to 0-3 of the backbone chain units. α-,β-, And γ-cyclodextrins, cyclic malto-oligosaccharides composed of 6, 7,and 8 glucose units were also not hydrolysed even after 24 h incubation.

The results obtained on dextran indicated that no α-(1→6) O-glycosidiclinkages could be cleaved by the product-specific amylase. The lack ofactivity on pullulan indicated that the product-specific amylase couldnot bypass α-(1→6) O-glycosidic linkages next to three glucose units orattack any of these three glucose units. The lack of hydrolysis on α-,β-, or γ-cyclodextrins indicated that the product-specific amylasehydrolysed starch by an exo-type of cleavage mechanism [30]. TheHPAEC-PAD trace (FIG. 6) also indicated a cleavage mechanism of theexo-type, since starch hydrolysis products larger than DP6 were absent.

To examine further the enzyme cleavage action on soluble starch, thestarch-iodine blue colour formation was plotted against the productionof reducing sugars (FIG. 7). The slope of the curve is indicating theprevalent type of cleavage mechanism of amylolytic starch hydrolysis[31]. An endo-acting enzyme will produce a slope with a smaller valuecompared to an exo-acting enzyme. A small value of the slope is theresult of a fast reduction of the starch-iodine blue colour complex dueto random amylolytic activity, indicated by the α-amylase from A. oryzae(the slope is −61). The extracellular enzyme preparation from P.stutzeri showed evidence for a prevalent exo-acting cleavage mechanismindicated by a larger slope value (the slope is −13). The purifiedproduct-specific amylase showed a slope value of −6, indicatingexo-activity.

The mode of action of hydrolysis of substrates with low DP by theproduct-specific amylase was examined by incubation with suchsubstrates. Maltose, maltotriose, and maltotetraose were not hydrolysedby the purified B. clausii BT-21 amylase. This confirms the resultsobtained on soluble starch, that these products are accumulating andtherefore considered as end products of the hydrolysis.

The product-specific amylase activity on a mixture ofmalto-oligosaccharides from DP4 to DP10 was studied by a time courseexperiment. The change of the peak areas obtained by the HPAEC-PADcorresponded to the formation or a hydrolysis of malto-oligosaccharides.The formation of maltohexaose (DP6) and the simultaneous decrease in theamount of DP7, DP8, DP9, and DP10 confirmed the maltohexaose formingability of the enzyme. However, steady state conditions were reached andthe further degradation of DP6 as found by starch hydrolysis was notdetected even after 7 days of hydrolysis. The concentration of DP6 wasmuch lower than the one obtained at the starch hydrolysis and indicatedthat a certain amount of maltohexaose was required for the formation ofmaltotetraose and maltose to proceed.

The starch hydrolysis by the B. clausii BT-21 product specific amylasewas found to resemble a two step procedure. This procedure included aninitial hydrolysis of starch to mainly maltohexaose and small amounts ofmaltopentaose, which were further hydrolysed to mainly maltotetraose andmaltose accumulating after extensive hydrolysis. The second hydrolysisstep to maltotetraose and maltose seemed to be limited by thepreliminary hydrolysis of the larger substrate to maltohexaose, since aconcentration dependence seemed to a regulator for the second step toproceed.

Baking Experiment

A baking experiment was performed with the product-specific amylase.Doughs were prepared with 10 g of standard Danish wheat flour (Danisco98078) and 6.2 ml 0.2 M NaOH-glycine buffer, pH 10 without (control) orwith 40 units of the enzyme (assayed at 45° C. and pH 10 as described inMaterials and Methods of Section B), baked and analysed by DSC afterstorage according to “Assays for measurement of retrogradation andstaling”. As shown in Table 8 the enzyme significantly reduces theamount of retrograded amylopectin found day 7 after baking whichindicates that it has a significant antistaling effect.

TABLE 8 Effect of B. clausii product-specific amylase on starchretrogradation based on measuring the melting enthalpy of retrogradedamylopectin (in J/g) 7 days after baking Multiple Range Tests forEnthalpy by Treatment Method: 95,0 percent LSD Treatment Count MeanEnzyme 16 2,44375 Control 16 2,56 Contrast Difference +/− LimitsControl - Enzyme *0,11625 0,0491094 *denotes a statistically significantdifference.

Crumb samples of the baked products frozen after the baking have beenextracted with distilled water (1 g baked product/10 g water, stirredfor 1 h and centrifuged) and analysed by HPAEC-PAD as described above todetect the starch hydrolysis products formed by the enzyme during thebaking of the doughs. Relative to the control accumulation ofmaltotetraose, maltopentaose, maltohexaose and maltoheptaose was foundas result of the activity of this enzyme.

Summary Section

The present invention discloses a process for making bakery products, aswell as amylases suitable for use in such a process.

Preferred embodiments of the present invention are now presented by wayof numbered paragraphs.

References

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1. A composition comprising a non-maltogenic exoamylase that hydrolysesstarch by cleaving off one or more linear malto-oligosaccharides,predominantly comprising from four to eight D-glucopyranosyl units, fromthe non-reducing ends of the side chains of amylopectin, together withat least one further dough ingredient or dough additive.
 2. Thecomposition of claim 1, in which the non-maltogenic exoamylase has anendoamylase activity of less than 0.5 endoamylase units (EAU) per unitof exoamylase activity.
 3. The composition of claim 1, in which thedough ingredient or dough additive comprises flour, in which the flouris wheat flour or rye flour or mixtures thereof.
 4. The composition ofclaim 1, in which the non-maltogenic exoamylase yields, in a waxy maizestarch incubation test, one or more hydrolysis products comprising oneor more linear malto-oligosaccharides of from one to tenD-glucopyranosyl units, and in which at least 60% by weight of thelinear malto-oligosaccharides of from one to ten D-glucopyranosyl unitsconsist of from three to eight D-glucopyranosyl units.
 5. Thecomposition of claim 4, in which at least 60% of the hydrolysis productis maltotetraose, maltopentaose, maltohexaose, maltoheptaose ormaltooctaose.
 6. The composition of claim 5, in which at least 60% ofthe hydrolysis product is maltotetraose.
 7. The composition of claim 6,in which the non-maltogenic exoamylase is obtained from Pseudomonassaccharophila.
 8. The composition of claim 7, in which thenon-maltogenic exoamylase is encoded by a DNA sequence comprisingGenBank accession number X16732.
 9. The composition of claim 5, in whichat least 60% of the hydrolysis product is maltohexaose.
 10. Thecomposition of claim 9, in which the non-maltogenic exoamylase isobtained from Bacillus clausii.
 11. The composition of claim 10, inwhich the non-maltogenic exoamylase has a molecular weight of about101,000 Da as estimated by sodium dodecyl sulphate polyacrylamideelectrophoresis.
 12. The composition of claim 11, in which thenon-maltogenic exoamylase has an optimum of activity at pH 9.5 and 55°C.
 13. The composition of claim 1, which further comprises a doughingredient or dough additive selected from the group consisting of doughstrengtheners and crumb softeners.
 14. The composition of claim 1, inwhich the dough ingredient or dough additive comprises an enzymeselected from the group consisting of proteases, oxidoreductases,glucose oxidase, hexose oxidase, ascorbate oxidase, hydrolases, lipases,esterases, glycosidases, amylolytic enzymes, α-amylase, pullulanase,xylanase, cellulose, and hemicellulase.
 15. An improver composition fora starch product, in which the composition comprises a non-maltogenicexoamylase that hydrolyses starch by cleaving off one or more linearmalto-oligosaccharides, predominantly comprising from four to eightD-glucopyranosyl units, from the non-reducing ends of the side chains ofamylopectin, together with at least one further dough ingredient ordough additive.
 16. The improver composition of claim 15, which thenon-maltogenic exoamylase is obtained from Pseudomonas saccharophila,and is encoded by a DNA sequence comprising GenBank accession numberX16732.
 17. The improver composition of claim 15, in which thenon-maltogenic exoamylase is obtained from Bacillus clausii, and has amolecular weight of about 101,000 Da as estimated by sodium dodecylsulphate polyacrylamide electrophoresis, and an optimum of activity atpH 9.5 and 55° C.
 18. A dough comprising a non-maltogenic exoamylasethat is capable of hydrolyzing starch by cleaving off one or more linearmalto-oligosaccharides, predominantly consisting of from four to eightD-glucopyranosyl units, from the non-reducing ends of the side chains ofamylopectin.
 19. The dough of claim 18, in which the non-maltogenicexoamylase is obtained from Pseudomonas saccharophila, and is encoded bya DNA sequence comprising GenBank accession number X16732.
 20. The doughof claim 18, in which the non-maltogenic exoamylase is obtained fromBacillus clausii, and has a molecular weight of about 101,000 Da asestimated by sodium dodecyl sulphate polyacrylamide electrophoresis, andan optimum of activity at pH 9.5 and 55° C.